Genetic heterogeneity in Prevotella intermedia, Prevotella nigrescens, Prevotella corporis and related species isolated from oral and nonoral sites

Prevotella intermedia (43 isolates), Prevotella nigrescens (55) and Prevotella corporis (8) from oral and nonoral sites were distinguished by species-specific DNA fragments, after hybridization of DNA fragments with ribosomal RNA (ribotyping). Eight strains previously identified as P. intermedia did not have these specific fragments. P. nigrescens, P. intermedia and P. corporis formed separate clusters in dendrograms constructed using clustering with an unweighted pair group method with arithmetic averages of similarity values derived from ribotype patterns, with 10 subclusters in P. intermedia isolates and 26 in P. nigrescens. Nine groups of P. intermedia isolates and 6 of P. nigrescens shared identical patterns. Specific ribotypes or species were not associated with particular diseases when all isolates were analyzed. However, results from organisms isolated by one laboratory using consistent clinical reporting indicated that P. intermedia was associated with more severe forms of periodontitis and P. nigrescens with mild to moderate disease.     

Hydrolytic and depolymerising enzyme activity of Prevotella intermedia and Prevotella nigrescens

OBJECTIVE: Prevotella intermedia has been reported to be associated with periodontal disease whilst P. nigrescens has predominantly been isolated from more specific conditions and healthy sites. The aim of the present study was to compare the enzyme activity of these species.
MATERIALS AND METHODS Nine strains of P. intermedio and 12 strains of P. nigrescens were studied. Lipolytic. saccharolytic, nucleolytic and proteolytic activity was determined by traditional microbiological and chromo-genic substrate methods.
RESULTS: All strains hydrolysed gelatine, casein. DNA and RNA. Lipase activity was produced by all strains except P. nigrescens ATCC 33563^T. Lipolytic activity of P. nigrescens strains decreased as the environmental glucose concentration was increased. Only two strains, both P. intermedia , hydrolysed benzyl-arg-p-nitroanilide. All strains hydrolysed alkaline pnitrophenolphosphate (except P. intermedia DAL 100). produced glycylprolyl dipeptidase activity and demonstrated elastase-like activity. All but three strains (2 P. intermedia and I P. nigrescens) hydrolysed suc-ala-ala-pro-phe-p-nitroanilide. Overall, no qualitatively analysed enzyme activity was exclusive to all strains of either species. Quantitatively analysed activity exhibited a high degree of variability both within and between species.
CONCLUSIONS: P. intermedia and P. nigrescens degrade natural and synthetic substrates, but intra- and interspec-ies activity is variable.     

中间普里沃菌基因多态性及其在成人牙周炎患者龄下菌斑中的分布

目的：了解中间普里沃菌（P．i)的基因多态性及其在成人牙周炎（AP）病变部位与健康部位的分布特点。方法：采用随机引物多聚酶链扩增法（APPCR）用法引物OPA－03和OPA－13对来自25例AP的101株P．i作基因型分析。结果：用引物OPA－03对101株P.i的APPCR分析,得到7种不同的基因型,用引物OPA－13对97株P.i的APPCR分析,得到12种不同的的基因型;同一患者口内有1－4种基因型,以1种为主,同一患者病变部位与健康部位均可有1－3种基因型,以1种为主;P.i在病变部位的优势基因型与P.i在健康部位的优势基因型基本一致,未发现与牙周健康状态相关的特定基因型P.i。结论：中间普里沃菌里内源性致病菌,在适宜的条件下过度生长导致牙周炎。     

中间普氏菌内毒素刺激人牙周膜细胞上调表达基因的研究

目的：用基因芯片研究中间普氏菌内毒素刺激前后，牙周膜细胞上调表达基因。方法：应用点样数512点的基因芯片分析中间普氏菌内毒素刺激人牙周膜细胞上调表达基因。结果：研究发现中间普氏菌内毒素刺激牙周膜细胞后有6条上调基因，其中部分基因与蛋白激酶和蛋白磷酸酶有关。结论：牙周致病菌内毒素刺激牙周膜细胞后，可引起部分基因表达上调，从而影响牙周膜细胞正常的生理功能。     

中间普氏菌内毒素刺激前后下调人牙周膜细胞表达基因的研究

目的 :研究中间普氏菌内毒素刺激前后牙周膜细胞下调表达基因。方法 :应用点样数 5 12点的基因芯片分析中间普氏菌内毒素刺激前后牙周膜细胞下调表达基因。结果 :研究发现中间普氏菌内毒素刺激牙周膜细胞后 8条下调基因 ,包括转录调控基因、凋亡相关基因和受体基因。结论 :中间普氏菌内毒素刺激牙周膜细胞后 ,可引起部分基因表达下调 ,从而影响牙周膜细胞正常的生理功能     

Similarity of salivary and subgingival Prevotella intermedia and Prevotella nigrescens isolates by arbitrarily primed polymerase chain reaction

The distribution and the genetic similarity of Prevotella intermedia and Prevotella nigrescens in saliva and in subgingival samples recovered from the same subject were studied in 16 subjects with different periodontal status. The isolates (4 salivary and 4 subgingival P. intermedia/nigrescens group isolates per subject) were identified to species level by hybridization with species-specific oligonucleotide probes, and the clonal analysis was performed using arbitrarily primed polymerase chain reaction (AP-PCR) (all isolates) and ribotyping (isolates from 5 subjects). In addition, the applicability of AP-PCR in differentiating between P. intermedia and P. nigrescens species was tested using 18 P. intermedia and 20 P. nigrescens isolates from 34 subjects. P. intermedia was detected in 7 and P. nigrescens in 14 of the 16 subjects. In all subjects the same species was found both in saliva and in subgingival plaque. In 15 of the 16 subjects, similar AP-PCR types of P. intermedia and/or P. nigrescens between salivary and subgingival samples were found. The salivary and subgingival isolates that were similar by AP-PCR were indistinguishable also by ribotyping. The AP-PCR analysis revealed a P. intermedia or P. nigrescens species-specific AP-PCR product in most isolates. This study indicates that both P. intermedia and P. nigrescens were found both in salivary and in subgingival samples, and both sampling sites within the same individual were usually colonized with identical AP-PCR types of the species. Thus, in addition to a subgingival sample a salivary sample seems to be suitable for detection and clonal analysis of these species. The AP-PCR method proved to be a simple method applicable for differentiation and clonal analysis of P. intermedia and P. nigrescens.     

Effects of lipopolysaccharide extracted from Prevotella intermedia on bone formation and on the release of osteolytic mediators by fetal mouse osteoblasts in vitro

Prevotella intermedia, a Gram-negative obligate anaerobic black-pigmented oral bacterium, belongs to a small group of microorganisms that is closely associated with the initiation of periodontal diseases. Lipopolysaccharide (LPS), an outer membrane component, is one of the main virulence factors of this bacterium. The aim of this study was to examine the effects of Prev. intermedia lipopolysaccharide, extracted by the hot-phenol-water method, on differentiation (alkaline phosphatase activity) and mineralisation (calcium incorporation) of fetal mouse calvarial cells in vitro and to determine the release of the important osteolytic factors nitric oxide, interleukin-6 (IL-6) and matrix metalloproteinases by these cells after treatment with different concentrations of Prev. intermedia lipopolysaccharide (0.2–25 μg/ml). By gelatin zymography, we also characterized the matrix metalloproteinases released by these osteoblasts. Treatment with Prev. intermedia lipopolysaccharide dose-dependently inhibited bone formation by reducing alkaline phosphatase activity and calcium incorporation and induced the release of nitric oxide, IL-6 and the latent proforms of MMP-2 and MMP-9 by fetal mouse osteoblasts in organoid culture. These results indicate that the lipopolysaccharide from Prev. intermedia not only participates in periodontal tissue destruction and alveolar bone resorption, but also inhibits bone formation.     

β-lactamase-producing strains in the species Prevotella intermedia and Prevotella nigrescens

A total of 96 strains were collected that included laboratory strains and clinical isolates classified Prevotella intermedia sensu lato and the type strains of the species P. intermedia sensu stricto and Prevotella nigrescens. Susceptibility to amoxicillin and amoxicillin-clavulanic acid was determined by the Etest. PCRDNA probe assays were used to speciate each strain as P. intermedia sensu stricto or P. nigrescens. By Etest, 71 strains (74%) were susceptible to both amoxicillin and amoxicillin-clavulanic acid with minimum inhibitory concentrations in the 0.016–0.064 μg/ml range. In contrast, amoxicillin minimum inhibitory concentrations of 25 strains (26%) were in the range of 1.5–96 μg/ml with concomitant amoxicillin-clavulanic acid minimum inhibitory concentrations in the low range 0.016–0.38 μg/ml, indicating a production of β-lactamase as confirmed by nitrocefin tests. Of these β-lactamase-producing strains, 20% (5/25) were identified as P. intermedia sensu stricto by the PCR-DNA probe assay and 72% (18/25) as P. nigrescens. Our results provide support for the major role of P. nigrescens in the failure of therapy using β-lactam antibiotics.     

The effects of tetracycline, minocycline, doxycycline and ofloxacin on Prevotella intermedia biofilm

Prevotella intermedia, a black-pigmented, anaerobic, gram-negative bacterium, is associated with various type of periodontitis. Antibiotic treatments via a systemic or local route have been reported as being useful for treating periodontal disease. The purpose of this study was to examine the effects of four antibiotics, tetracycline (TET), minocycline (MINO), doxycycline (DOXY) and ofloxacin (OFLX) on P. intermedia biofilms at minimum inhibitory concentrations (MIC) from one-fold to 100-fold. MICs were determined for planktonic cells. Biofilm formation was determined with the crystal violet stain method and the bioactivities in the biofilms were determined with the adenosine triphosphate (ATP) -bioluminescent assay using a 96-well culture plate. At one-fold MIC, DOXY inhibited biofilm formation by P. intermedia ATCC 25611. Other antibiotics at one-fold MIC had no effects on the biofilm formation of tested bacterial strains. In P. intermedia ATCC 25611 biofilms, all the antibiotics tested showed inhibitory activities at five- to 100-fold MICs. In the biofilms of P. intermedia strains, except ATCC 25611, treated with three tetracycline antibiotics, the bioactivities were significantly increased, indicating the difficulties involved in designing antibiotic therapy for periodontal disease.     

The microbial synergy of Peptostreptococcus micros and Prevotella intermedia in a murine abscess model

This study characterized the microbial interaction of Peptostreptococcus micros and Prevotella intermedia, the major pathogens of dentoalveolar infection, using a murine model. Subcutaneous injection of P. micros cells in the dorsum of the mouse together with living cells of P. intermedia resulted in a significantly larger abscess when compared with single injection of the organisms (P < 0.02). The abscess size was also significantly increased (P < 0.05) when the plate-cultured cell suspension of P. micros was injected into mouse with the culture filtrate of P. intermedia. The heat-treated culture filtrate of P. intermedia also enhanced the virulence of P. micros. P. micros culture filtrate did not affect the virulence of P. intermedia. Interestingly, the virulence of P. micros appeared to be enhanced even when the culture filtrate of P. intermedia was injected at separate sites in the mouse. These results suggest that a heat-stable product or products of P. intermedia increase the virulence of P. micros indirectly by altering the host condition, whereas living cells of P. micros can directly enhance virulence of P. intermedia.     

Pathogenicity of Prevotella intermedia and Prevotella nigrescens isolates in a wound chamber model in rabbits

The pathogenicity of 14 isolates identified as Prevotella intermedia or Prevotella nigrescens by serogrouping using monoclonal antibodies was compared in a tissue cage model in rabbits. Seven strains from periodontal abscesses, 5 strains from deep periodontal pockets and 2 strains from gingivitis were tested in the animal model comprising 6 Teflon tissue cages implanted on the back each of 34 rabbits. A total of 105–108 cells of P. intermedia or P. nigrescens strains were inoculated alone or together with either Actinobacillus actinomycetemcomitans or Streptococcus mitis. Five strains of Porphyromonas gingivalis were used as a reference. The infectivity was recorded as pus formation and log viable count in aspirated material for 3, 7 and 14 days. None of the Prevotella strains inoculated in monoculture survived more than 3 days, and they had no capacity to produce abscess. P. intermedia or P. nigrescens strains in combination with A. actinomycetemcomitans produced abscesses in 33–100% and with S. mitis in 42–100%. No difference in abscess formation or log viable count in samples after 14 days was recorded between serogroup I ( P. intermedia ) and serogroup II and III ( P. nigrescens ). The infectivity of P. intermedia or P. nigrescens strains did not differ whether they were isolated from periodontal abscess, periodontal pocket or gingivitis. P. intermedia and P. nigrescens strains produced abscesses in combination with a facultative anaerobic strain and appears to have a similar pathogenicity in the wound chamber model in rabbits.     

中间普氏菌在家庭成员牙周菌斑中的分布调查

目的：调查了解中间普氏菌在牙周健康人群中的分布状况。方法：收集符合纳入标准的６０个家庭、１８１例受试者的牙周颈缘龈上菌斑和龈下菌班,采用厌氧菌培养获得２７９株产黑色素的Ｇ＾－厌氧杆菌,然后进行产黑色素的Ｇ＾－厌氧杆菌的纯化培养及微量生化鉴定。结果：中间普氏菌在牙周较健康的父母和儿童牙周菌班中均可检出,儿童群体中中间普氏菌阳性率达７０．４９％,而成人群体中中间普氏菌阳性率为４３．３３％,二者有显著性     

牙龈卟啉菌、中间普氏菌的分离、培养和鉴定

目的：采用厌氧培养和鉴定技术，分离牙周炎患者龈下菌斑中的牙龈卟啉菌和中间普氏菌。方法：采集牙周炎患者的龈下菌斑，厌氧培养，分离产黑色素菌。经长波长紫外灯观察，各种生化分析和间接免疫荧光染色，分离和鉴定牙龈卟啉菌，中间普氏菌。结果：在受检的33例牙周炎患者中，24例患者检出了牙龈卟啉菌，总检出率为72．77％，共分离了79株。18例患者检出了中间普氏菌，总检出率为54．54％。共分离了32株。结论：厌氧培养，生化反应鉴定技术的发展，使牙龈卟啉菌和中间普氏菌的分离与培养准确可靠，为今后在分子水平上了解各菌株的致病机理打下了基础。     

β-lactamase-producing strains in the species Prevotella intermedia and Prevotella nigrescens

A total of 96 strains were collected that included laboratory strains and clinical isolates classified Prevotella intermedia sensu lato and the type strains of the species P. intermedia sensu stricto and Prevotella nigrescens. Susceptibility to amoxicillin and amoxicillin-clavulanic acid was determined by the Etest. PCRDNA probe assays were used to speciate each strain as P. intermedia sensu stricto or P. nigrescens. By Etest, 71 strains (74%) were susceptible to both amoxicillin and amoxicillin-clavulanic acid with minimum inhibitory concentrations in the 0.016–0.064 μg/ml range. In contrast, amoxicillin minimum inhibitory concentrations of 25 strains (26%) were in the range of 1.5–96 μg/ml with concomitant amoxicillin-clavulanic acid minimum inhibitory concentrations in the low range 0.016–0.38 μg/ml, indicating a production of β-lactamase as confirmed by nitrocefin tests. Of these β-lactamase-producing strains, 20% (5/25) were identified as P. intermedia sensu stricto by the PCR-DNA probe assay and 72% (18/25) as P. nigrescens. Our results provide support for the major role of P. nigrescens in the failure of therapy using β-lactam antibiotics.     

The Prevotella intermedia group organisms in young children and their mothers as related to maternal periodontal status

Currently, the Prevotella intermedia group includes three biochemically and phylogenetically related species: Prevotella intermedia, Prevotella nigrescens, and the newly described Prevotella pallens. The two first-named species are mentioned with varying emphasis in connection with periodontal diseases, while such a connection of P. pallens is not known. Mothers serve as a plausible source of bacteria to their children, and conceivably, a mother with periodontitis as a recurrent reservoir of periodontally infecting organisms. In the present study, 23 mothers and their young children were examined for the presence of the P. intermedia group organisms in relation to maternal periodontal status (I: periodontal health, II: initial periodontitis, and III: advanced periodontitis). Species differentiation was based on established biochemical methods, electrophoretic mobility patterns, SDS-PAGE, and DNA hybridization. P. intermedia was not recovered from children but nearly exclusively from mothers in group III, thus confirming its association with periodontitis. P. nigrescens and P. pallens were frequently found in mothers and children. To determine bacterial transmission between a mother and her child, 72 isolates from 13 mother-child pairs were analyzed by arbitrarily primed PCR (AP-PCR). Similar AP-PCR types of P. nigrescens and/or P. pallens were recovered from 3/4 pairs in group I, 2/5 pairs in group II, and none in group III. Our results indicate that different species within the P. intermedia group have a different colonization pattern in childhood and that the periodontal status reflects qualitatively their presence in maternal saliva. Intra-familial transmission of P. nigrescens and P. pallens can occur in early childhood, however similar AP-PCR types were most obvious within periodontally healthy mother-child pairs.     

中间普氏菌内毒素对人牙周膜细胞增殖的时间效应和细胞周期的影响

目的 :观察中间普氏菌内毒素 (Pi)对离体培养的人牙周膜细胞增殖的时间效应和细胞周期的影响。方法 :采用细胞培养技术、噻唑盐比色测定法 (MTT)和流式细胞仪技术。结果 :MTT法结果表明 ,加入10 μg/mLPi内毒素 3h后 ,即明显抑制人牙周膜细胞的增殖 (P <0 .0 5 ) ,且抑制作用随时间的延长而增强。流式细胞仪结果表明 ,在 10 μg/mLPi内毒素作用后 6和 12h ,DNA合成前期细胞所占百分比 (G1% )有所增高 ,而DNA合成期细胞所占百分比 (S % )降低。作用 2 4h后S期细胞数量增加 ,G1期细胞数量下降。结论 :Pi内毒素对人PDLC有直接毒害作用 ,可能是内毒素抑制静止态的G1期细胞进入S期 ,从而抑制细胞的增殖。     

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目的应用聚合酶链式反应-变形梯度凝胶电泳（PCR—DGGE）技术检测慢性根尖周炎患牙根管内中间普菌和链球菌定植情况,分析根管内细菌与患牙症状间关系。方法2011年12月至2013年5月于北京大学深圳医院口腔科就诊的27例慢性根尖周炎患牙根管内细菌样本,提取DNA,利用16SrDNA引物进行PCR—DGGE技术分析。结果27例共检出细菌菌属17种。中间普菌在17例有症状组中检测出16例（94．1％）,在10例无症状组中检测出6例（60．0％）,两组检出率差异有统计学意义（P〈0．05）。链球菌在17例有症状组中均未检测出,在10例无症状组中检测出4例（40．0％）,两组检出率差异有统计学意义（P〈0．05）。结论慢性根尖周炎患牙以厌氧菌感染为主,根管内中间普菌、链球菌与临床症状相关。